Muscular dystrophy represents a family of inherited diseases of the muscles. Some forms affect children (e.g., Duchenne dystrophy) and are lethal within two to three decades. Other forms present in adult life and are more slowly progressive. The genes for several dystrophies have been identified, including Duchenne dystrophy (caused by mutations in the dystrophin gene) and the teenage and adult onset Miyoshi dystrophy or its variant, limb girdle dystrophy 2B or LGMD-2B (caused by mutations in the dysferlin gene). These are “loss of function” mutations that prevent expression of the relevant protein in muscle and thereby cause muscle dysfunction. Mouse models for these mutations exist, either arising spontaneously in nature or generated by inactivation or deletion of the relevant genes. These models are useful for testing therapies that might replace the missing protein in muscle and restore normal muscle function.
Differentiated muscle is composed of multi-nucleated cells or myofibers that have an extraordinary capacity to regenerate. This regenerative capacity exists because muscle possesses primitive muscle precursor cells (muscle stem cells and somewhat more mature cells known as “satellite cells”). These cells lie dormant in muscle and can be activated to make new mononucleated muscle cells (myoblasts) that can adhere to one another and fuse to make new, multi-nucleated myotubes, as well as the more mature muscle cells (that are again multinucleated). Because myofibers arise from the fusion of individual myoblasts, a protein made by one muscle cell is readily accessible to be shared with neighboring muscle cells lacking that protein if the two cells fuse into the same myotube.
Inherent in this concept of myoblast fusion and muscle regeneration is the possibility of cell therapy of muscle diseases. The fusion of a myoblast capable of making a muscular dystrophy protein with muscle cells that lack the protein should correct the deficiency in the resulting myotube. That is, the normal nucleus in the normal myoblasts replaces a gene missing in the dystrophic muscle cells thus achieving gene and protein replacement through cell therapy.
Partridge and colleagues demonstrated more than a decade ago that a mixed population of muscle precursor cells capable of making normal dystrophin protein could fuse into muscle of the mdx mouse that lacks dystrophin and thereby partially replace the missing protein (Partridge et al., Nature 337:176-179, 1989). In the seminal experiments of Partridge, it was not clear precisely what populations of the muscle precursor cells had the capacity to achieve this effect. At least six human trials of myoblast therapy were undertaken in Duchenne and Becker dystrophy patients, using direct intramuscular injections of myoblasts; that none were effective might be interpreted to mean that myoblasts were not sufficiently undifferentiated to participate effectively in muscle cell therapy. This observation stimulated the search for muscle stem cells.
Over the last two years, several muscle biologists have had encouraging initial success in isolating putative muscle stem cells. Moreover, these studies have documented not only that the primitive muscle precursors can fuse into injured muscle to make new muscle, but also that such stem cells or stem-like cells have an extraordinary capacity to circulate in the blood and then to leave the blood to enter sites of focal muscle injury in response to unidentified myotropic factors. Strikingly, in the last three years it has become apparent that cells with features of muscle stem cells may be present in tissues thought previously to be primarily hematogenic, such as the bone marrow. In 1999, Gussoni and colleagues in the Kunkel laboratory reported that a population of primitive cells identified by the presence of a multi-drug resistance transporter as a “side population”(SP) fraction of cells in either the bone marrow or muscle itself could be delivered to dystrophic mdx muscle following tail vein injection (Gussoni et al., Nature 401:390-394, 1999). That these cells included primitive stem cells was strongly suggested by the finding that the same injection could populate muscle tissue with enough normal muscle cells to restore dystrophin expression in up to 10% of myofibers and, at the same, repopulate the bone marrow of previously irradiated recipient mice. Analogous findings were subsequently reported from the laboratories of Huard (Lee et al., J. Cell Biol. 150:1085-1099, 2000) and Bresolin (Torrente et al., J. Cell Biol. 152:335-348, 2001).
At this time, one criterion for defining a cell as a muscle stem cell is the capacity to differentiate to form myoblasts and thereby augment some aspect of muscle regeneration or repair. Typically, the expression of a previously missing protein (like dystrophin) after muscle stem cell infusion provides prima facie evidence that a muscle stem cell is present. To date, no single set of molecules have been identified that uniquely define muscle stem cells. However, an evolving family of surface proteins is being identified that characterizes different stages of differentiation in the muscle cell lineage, as summarized in Table 1.
TABLE 1Selected Surface Markers Differentially Expressedon Cells in the Muscle LineageSatelliteMPC/MarkerMSC/SPcellMyoblastMyotubeSca-1+—c-kit− (?+)+CD34++CD45 (heme−−lines)Desmin−+bcl-2?+Flk-1− (human)(VEGFR/kdr)M-cadherin−+Pax-7? +/−+c-met receptor?+Multidrug res+?protMyoD−−++Myogenin−−++MRF4−−++NCAM−−+++CD43−−−−CD123−CD90 (+ on MP)+AC133−/+?MSC = muscle stem cell; SP = side population cells; MPC = muscle precursor cellsFor example, it is likely that a candidate set of stem cells will express the surface antigen CD34 and perhaps other primitive cell surface markers such as AC133, but not lineage markers, such as c-kit or the hematopoietic marker CD45.
Umbilical cord blood cells (“UCB” cells) contain sub-populations with properties of stem cells. Replacement of bone marrow cells can be accomplished via infusion of human UCB cells.